Our goal is to characterize novel non-18-carbon, L-threo-sphinganines to act as synergizing agents for the dihydroceramide-increasing, cytotoxic retinoid, fenretinide (4-HPR). Ceramides are sphingolipids with pro- death signaling properties. All naturally-occurring mammalian dihydroceramides and ceramides utilize 18- carbon (C18) D-erythro-sphinganine/sphingosine backbones, excepting central brain tissues, in which half of ceramides are made on 20-carbon (C20) D-erythro-sphinganine/sphingosine. We have previously reported that 4-HPR selectively-increased dihydroceramides in cancer cells and that 4-HPR cytotoxicity was markedly synergized by the artificial sphinganine variant, (C18)-L-threo-sphinganine (safingol). We have made the unexpected observation that certain other artificial, L-threo-, non-(C18) carbon chain length sphinganines, specifically C17-, C19- and C20-L-threo-sphinganines, but not (C12)-L-threo-sphinganine, are metabolized to L-threo-dihydroceramides in human cancer cells, resulting in a multi-log synergistic enhancement of 4-HPR cytotoxicity. Using a novel, NCI RAID-supported, high-dose intravenous 4-HPR formulation, we recently demonstrated multiple, sustained Complete Responses in relapsed T-cell lymphomas in a Phase I trial. This intravenous formulation is currently moving forward into Phase II trials. We hypothesize that individual non- (C18)-L-threo-sphinganines will have greater synergistic cytotoxicity with 4-HPR than safingol in certain cancer types with less systemic toxicity. Our preliminary data suggest that the synergistic cytotoxicity of these L-threo- sphinganines derives from their metabolism to L-threo-dihydroceramides concurrent with the increase of native long acyl chain dihydroceramides driven by high-dose fenretinide in susceptible cancer cells. Our Specific Aims are: AIM 1. Determine the cell penetrance and metabolism vs. chain length of non-(C18)-L-threo- sphinganines under hypoxic conditions using C14 radiolabeling, thin layer chromatography and mass spectrometry; AIM 2. Determine the cytotoxicity vs. chain length profile for non-(C18)-L-threo-sphinganines + 4-HPR in a cell line panel of normal human cells and different cancer types with an emphasis on neural tumors, using fixed ratio, dose-response cytotoxicity assays in 2% oxygen and Combination Index analysis; determine the mechanistic mode of cell death. AIM 3. Determine if the synergistic cytotoxicity of non-(C18)-L- threo-sphinganines is dependent on a concurrent increase of native dihydroceramides by combining supplemental sphinganine with concurrent dihydroceramide synthase inhibition to mimic the action of 4-HPR in cancer cells. These studies may identify cancer-type specific agents that synergize the cytotoxicity of dihydroceramide-increasing chemotherapy agents. PUBLIC HEALTH RELEVANCE: We will determine if certain novel artificial analogs of sphinganine (called, L-threo-sphinganines) can increase the anti-cancer activity of the vitamin-A derivative, fenretinide (4-HPR), in a cancer cell type-specific manner. If so, they will be candidates for future translational studies in combination with our novel intravenous fenretinide formulation, which is currently moving into Phase 2 trials.